Genome Sequence Acquisition and Analysis

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Genome Sequence Acquisition and Analysis
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Outline

• Defining Genomes
• What Have We Learned from the Human Genome Draft
  Sequences?

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Goals for this Chapter 1

• 1.1 Defining Genomes
• Define the filed of genomics
• Learn how genomes are sequenced
• Understand the utility of short DNA segments
• Utilize online tools to analyze genome sequences

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What Is Genomics?

• Genome
• The total DNA content of a haploid cell or half
  the DNA content of a diploid cell.
• Genomics
• Genomics involves large data sets (about 3
  billion base pairs for the human genome) and
  high-throughput methods (fast methods for
  collecting the data)
• Sequencing DNA and collecting genome variations
  with a population as well as transcription
  control of genes.
• From DNA sequence analysis to an organisms
  response to environmental perturbations.

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Proteomics

• Proteome
• The complete protein content of a cell/organism
  at a given moment.
• Proteomics (-omic)
• Other terms
• Transcriptome
• Metabolome

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How Are Whole Genomes Sequenced?

• Sanger method (dideoxy method)
• Dr. Fred Sanger
• Polymerase chain reaction (PCR)

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Figure 1.1
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Figure 1.2
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What Is An E-value?

• A BLASTn search returns hits, sequences that
  produce significant alignments to the query
  sequence.
• The significance of a hit is measured by its
  E-value, or expect value.
• Biological significance hits will tend to have
  E-values much less than 1.0.
• The larger the E-value, the greater the chance
  that the similarity between the hit and the query
  is due to more coincidence.

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Why Do the Databases Contain So Many Partial
Sequences?

• Human Genome Project (HGP)
• By comparing different genomes, we should be able
  to better understand genomes in general, and the
  human genome in particular.
• Sequence-tagged Sites (STSs)
• The short segments of unique DNA sequence along
  every chromosome.
• Bacterial Artificial Chromosome (BACs)
• 150 Kb
• Yeast Artificial Chromosome (YACs)
• 150 Kb 1.5 Mb

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Figure 1.3
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Expressed Sequence Tags (ESTs)

• The short segments of cDNA
• Used to identify genes
• ESTs hints at the size of the genome and
  alternative ways to splice mRNA.
• ESTs is helpful for labs interested in cloning
  particular genes.
• Related database
• dbEST,
• UniGene,
• HomoloGene.

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Institutes

• National Institutes of Health (NIH)
• The Institute for Genomics Research (TIGR)

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How Do We Make Sense of All These Bases?

• Online Mendelian Inheritance in Man (OMIM)
• A comprehensive list of everything known about
  human biology and diseases.
• GeneCard Database of annotated genes in human
  genome.
• ORFs and Translation
• Open reading frames (ORFs, pronounced orphan)
• Recorded as accession number.
• Coding Sequence (CDS)

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Can We Predict Protein Functions?

• Kyte-Doolittle plot (hydropathy plot)
• To predict whether a protein is an internal
  membrane protein or not.
• The 3D shape of a protein is probably its most
  important characteristics.
• Conserved Domain (CD)

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3D Structures

• Protein Data Banks (PDB)
• Entrez Structure

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Structure-Function Relationships

• Gene Ontology (GO)
• Biological process is the why the overall
  objective toward which this protein contributes.
• Molecular function is the what the biochemical
  activity the protein accomplishes.
• Cellular component is the where the location of
  protein activity.
• Gene Ontologys unification of protein roles will
  help us communicate more effectively as we
  determine how genomes produce multifunctional
  cells.

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How Well Are Genes Conserved in Diverse Species?

• Clusters of Orthologous Groups (COGs)
• Enzyme Commission (EC) numbers
• Swiss-Prot
• Phylogenetic tress
• Paralogs
• The genes arose from a common ancestral gene
  within one species
• Orthologs
• The same gene in two organisms evolved from a
  common ancestral gene in another species.
• Synteny
• Genetic loci located on the same chromosome
  within a species, even if they ware separated by
  a great distance.
• Homology
• Two sequences were described as homologous if
  their sequences were similar because of a common
  evolutionary origin.

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How Do You Know Which Bases Form a Gene?

• Intergenic sequence
• Eukaryotic genes may contain introns as well as
  the coding exons.
• The ORFs are called pseudogenes since mutation
  has rendered them nonfunctional.

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Gene Expression Process
(???)
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The Gene Structure
Genes
Coding Regions
Upstreams
Downstreams
DNA-binding
Transcription Factors
5-AGCAATAGG-3 3-TCGTTATCC-5
Binding Sites
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Transcription Factors (TFs)

• DNA-binding proteins
• Recognize specific sites (sequences) gt binding
  sites.
• Transcription factors activate transcription
  initiation by RNA Polymerase II or III.
• Regulating the gene transcription process.

Binding Site
A Transcription Factor
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Some TF Binding Sites
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Intron and Exon
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RNA Splicing (Pre-mRNA -gt Mature mRNA)
Gene
5
3
DNA
Transcription
Exon
Intron
Transcription
pre-mRNA
Splicing
RNA
mRNA
Translation
Translation
Protein
protein
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Splice Sites
Exon
Exon
5
3
Intron
?
?
?
Pre-mRNA
Cut
Cut
PyPyPyPyPyNCAG?
AG?GTAGGT
Donor site (5 splice site)
Acceptor site (3 splice site)
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How Many Proteins Can One Gene Make?
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Goals for this Chapter 2

• 1.2 What Have We Learned from the Human Genome
  Draft Sequences?
• Survey human genome
• Verify genome annotations with online tools
• Recognize alternative forms of genes
• Explore epigenetic regulation of genome function

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Overview of Human Genome First Draft

• Published on 15 Feb. 2001.
• Humans have approximately 35,000 genes.
• Draft sequence means the DNA was sequenced on
  average four times, with finished sequence having
  eightfold coverage and errors estimated to be one
  in 10,000 bp.

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Figure 1.5
CpG dinucleotides from CpG island and the
cytosine base is often methylated.
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UTR
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Figure 1.6
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3-Dimensional
Gene Regulation Mechanism
TF 1
TF 2
Cooperation (Protein-Protein Interaction)
Correlation of Site Occurrences
Binding Site 2
Binding Site 1
Transcription start direction (??????)
The graphic is from Dr. Thomas Werners tutorial
in ISMB2000.
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Figure 1.7

• Humans have more cells with specialized functions
  than yeast, or worms.
• Human needs to regulate gene expression very
  carefully.
• Proteome complexity is regulated by gene
  expression.

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Figure 1.8a
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Figure 1.8b
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When Are the Data Sufficient?

• A Gene Is a Gene Is a Gene Short of
• Every Gene Has a Promoter
• Other Than rRNA and tRNA, All Genes Produce
  Proteins

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Can the Genome Alter Gene Expression Without
Changing the DNA Sequence?

• Imprinting, a process mammals use to mark a small
  set of genes during gametogenesis so that only
  the paternal (??) or maternal (??) copy will be
  transcribed and the other allele at the same
  locus will remain silent.
• As of 2002, there are more than 20 mammalian
  genes are known to be imprinted.

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What Is the Fifth Base in DNA?

• Methyl-Cytosine
• Dnmt1p is an enzyme that adds a methyl (CH3)
  group onto hundreds of thousands but not all
  cytosines throughout the genome.
• Methylome is a significant component of gene
  regulation and thus the proteome.
• Direct methylation may block the transcription of
  some genes.

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Figure 1.11